The increased ability to analyze the genetic makeup of individuals from different species has allowed a number of new techniques to be developed. Detection of genetic polymorphisms is useful for differentiating between individuals, parentage testing, forensic testing, analysis of relatedness of individuals, and mapping genes of interest linked to the microsatellite repeats.
Microsatellites are perfect, imperfect, or compound arrays of tandemly repeated nucleotide sequences embedded in an otherwise unique nucleotide sequence. Microsatellite repeats typically range from one to six base pairs (bp) in length. The microsatellite repeat arrays vary in the number of repeats from 6 to 30 or more in humans. However, the longer arrays of repeats are less frequently isolated. Microsatellites may consist of simple repeats containing only one uninterrupted repeated sequence, imperfect repeats containing two identical repeats separated by a small interval of non-repeated nucleotides, or compound repeats containing several different repeated sequence types (Weber, 1990). For an individual, any particular microsatellite chromosomal locus may vary in the number of repeats present.
Commonly used methods of genetic mapping by microsatellites take advantage of length variations among individuals (Weber and May, 1989). Utilizing the nucleotide sequence of the cloned microsatellite and its flanking regions, oligonucleotide primers for the PCR are designed that anneal to unique sequences that flank the repeats. The primers can be designed as near or far from the microsatellite as desired, the only limit being the resulting size of the PCR product for subsequent analysis.
Detection and size determination of PCR products from a specific microsatellite locus can be accomplished by several means. As the procedure was first described, PCR products are labeled with .sup.32 P, fractionated by a denaturing polyacrylamide gel electrophoresis and visualized by autoradiography. Alternatively, the PCR products are labeled with a fluorochrome, and separated on an automated DNA sequencing apparatus. Another method separates the PCR products by capillary electrophoresis, which has the advantage of being much faster than acrylamide gel electrophoresis while maintaining the accuracy of sizing (Buttler et al., infra.).
Whichever method is used for sizing of the DNA fragments, there is a limit to the resolution that is achieved. It becomes more difficult to obtain resolution of 1-2 bp when the fragments sized are greater than 500 bp, since the size difference between the products as a percentage of the total fragment size is small. Therefore, PCR primers must be designed such that the products of the reaction are easily distinguished from each other and accurately sized. Primers are usually designed to generate PCR products of 50 to 500 bp. In the future, methods may be developed to accurately resolve fragments of 1 Kb or even greater.
To date, many polymorphic dinucleotide microsatellites, usually of the (CA)n motif, have been isolated from the canine genome (Holmes et al., 1993; Ostrander et al., 1993). However, few polymorphic microsatellites other than those based on dinucleotide motifs have been isolated. There are several difficulties associated with the use of microsatellites based on dinucleotide motifs. One problem is the inherent difficulty of reproducible sizing, due to the high resolution required to accurately determine the size of PCR products derived from dinucleotide repeats. Another obstacle is the presence of so-called "stutter" bands generated due to slipped-strand mispairing during PCR, which is especially noticeable when employing microsatellites with dinucleotide motifs. These artifactual bands, which appear at 2 bp intervals below the correct size band, can make determination of allele size difficult in many cases.
Based on the inherent difficulties in typing with dinucleotide repeats, there is interest in determining trinucleotide and tetranucleotide repeat polymorphisms. Characterization of such polymorphisms may be useful in automation and standardized genotyping.